The articles set forth in the Background of the Invention are each incorporated herein by reference.
Mammalian proteins present in clinical samples (e.g. whole blood, serum, plasma, cerebrospinal fluid, and urine) are useful as indicators of a disease state or a bodily condition. The amount and type of these proteins in the sample can provide a wealth of information to the clinician.
For example, the protein components of serum include albumin, alpha-1 lipoprotein, alpha-2 macroglobulin, beta-1 lipoprotein and immunoglobulins (including gammaglobulins). Albumin, the major protein of serum, is usually present in a concentration of between 4.0 and 5.0 g/dL. Decreased concentration of albumin can be indicative of renal disease; increased concentration of albumin is characteristic of dehydration. Elevated levels of alpha-1 lipoprotein can be indicative of chronic alcoholism or hyperestrogenism due to, e.g., pregnancy. Elevated levels of beta-1 lipoprotein can be indicative of increased cholesterol levels.
Mammalian proteins are charged proteins containing both cationic and anionic moieties. They thus lend themselves to analysis by capillary zone electrophoresis ("CZE"). CZE is a technique which permits rapid and efficient separations of charged substances. In general terms, CZE involves introduction of a sample into a capillary tube and the application of an electric field to the tube. The electric field pulls the sample through the tube and separates it into its constituent parts. I.e., each of the sample constituents has its own electrophoretic mobility; those having greater mobility travel through the capillary faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during the migration of the sample through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones. The detector measures the absorbance of light by each constituent at a specified wavelength; different constituents absorb light differently, and, because of this, the constituents can be differentiated from each other.
CZE can be generally separated into two categories based upon the contents of the capillary columns. In "gel" CZE, the capillary tube is filled with a suitable gel, e.g. polyacrylamide gel. Separation of the constituents in the sample is predicated in part by the size and charge of the constituents travelling through the gel matrix. In "open-tube" CZE, the capillary tube is filled with an electrically conductive buffer solution. Upon application of an electric field to the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution must flow in this direction to maintain electroneutrality. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. The buffer in open CZE is as stable against conduction and diffusion as the gels utilized in gel CZE. Accordingly, separations can be obtained in open CZE quite similar to those obtained in gel-based electrophoresis.
Typically, the pH of the buffers utilized in open CZE are chosen with reference to the isoelectric points (pI) of the constituents in the sample. For example, the pI of serum albumin is 4.6; therefore, at pH 4.6, negatively charged and positively charged moieties of serum albumin are equal and the overall charge is neutral. However, as the pH is raised above the isoelectric point, the negatively charged moieties predominate and the net charge is negative. Thus, by selection of the proper pH, all of the species of the sample will be negatively charged. For serum samples, at pH greater than about 8.00, the majority of all serum-protein species will be negatively charged. Thus, manipulation of the isoelectric points of sample species can be used to ensure a proper charge distribution vis-a-vis the flow of such species through a charged capillary.
Typically, the results of CZE analysis are provided via an electropherogram which depicts the discrete zones of the sample constituents as peaks of various height and width. Additionally, the results can be presented in terms of numerical data based upon the integrated area under each constituent peak.
A problem encountered with capillary zone electrophoresis of samples is that the same sample constituents may appear on the electropherogram at different migration times for different samples. Stated again, a constituent common to two different samples may show up at a different place on each of the electropherograms for such samples. This is due, in part, to the fact that the amount of time taken by each earlier sample constituent as it passes through the capillary will affect the migration time of latter sample constituents.
In order to identify the specific constituent species in the sample being analyzed, the particular shape and location of a constituent species' electropherogram peak, relative to other constituent peaks, is typically determined. Stated again, the identification of a constituent species is based upon the particular electropherogram peak generated by that species. Alternatively, identification of the constituent species is based upon the retention time of that particular species. "Retention time" is defined as the period from initiation of analysis of the sample to detection of a sample constituent by the on-line detector.
However, skill is required in visually identifying the electropherogram peaks of particular constituents or identifying a constituent species based upon its retention time. For example, gammaglobulin, beta-1 lipoprotein, alpha-1 lipoprotein, and alpha-2 macroglobulin, which are serum proteins, all have about the same retention time and about the same electropherogram peak sizes when present in normal concentrations.
Furthermore, the concentration of one species in a sample can effect the shape and location of the electropherogram peak of another species in that sample. Additionally, as the concentration of a particular constituent species changes from sample to sample, so too will the retention time of that particular species. This can hamper identification of the individual constituents because a particular constituent will not always appear at the same electropherogram location from sample to sample.
In situations where different samples obtained from the same source but at different times may evidence different constituent concentrations, or in situations where samples obtained from different sources have the same constituents but include different concentrations for these constituents, it is essential that efficient, rapid and reliable identification of the sample constituents can be made without resort to visual identification guess-work or concern as to changes in the sample which will affect the retention times of the sample constituents.